A New Species of Mayfly (Ephemeroptera: Trichorythidae) from Mindanao Island, Philippines and Association of Life Stages Using DNA Barcode*

ARTICLE | Philippine Journal of Systematic Biology

Leocris S. Batucan Jr.1,2, Olga M. Nuñeza2, Reagan Joseph T. Villanueva3 and Chung-Ping Lin1,4

ABSTRACT KEY WORDS : A new mayfly species, Sparsorythus buntawensis sp. nov. (Tricorythidae) from Mt. Malindang Layawan River of Mt. Malindang in Mindanao Island, the Philippines is described based Macroinvertebrate on nymphal and adult morphologies. Sparsorythus buntawensis sp. nov. differ from all Sparsorythus known members of the genus primarily in the deeper cleft of the hypopharynx and wider Freshwater distance between compound eyes. Conspecific specimens of various life stages and Layawan River sexes of this new mayfly were associated using DNA barcode.

INTRODUCTION Mindanao Island of the Philippines, a previously unknown mayfly species have been discovered, a new Sparsorythus The species diversity of mayflies (Insecta: Ephemeroptera) in (Tricorythidae). The Tricorythidae are widespread in South- the Philippines, like in the whole Oriental region, is largely east Asia and the Indian subcontinent, and currently underestimated due to insufficient collecting effort and the encompass eight described species (Barber-James et al., region being largely “unknown lands” (Barber-James et al., 2013) within the genus Sparsorythus. Recently revised by 2008). Hubbard & Pescador (1978), cataloguing only 20 Sroka & Soldán (2008) based on Ulmer’s (1913) Philippine mayfly species, suggested that the low species Tricorythus jacobsoni from Java Island, Sparsorythus is number in the archipelago did not necessarily reflect a low considered as phylogenetically derived within the species diversity but may be caused by sporadic and Tricorythidae due to its morphological apomorphies. inadequate taxonomic works. From the first collecting record Sparsorythus differs from other tricorythids by its reduced made by Walker (1853) in Luzon to the most recent maxillary palps in nymphs, fully merged penial lobes publication of five new species by Braasch (2011) in Palawan, without auxiliary processes, and a well–developed only a total of 37 mayfly species were described from the paracercus in the imagines (Sroka & Soldán, 2008). Philippine archipelago. They are classified into eight families: Ulmer’s (1924) record of Sparsorythus jacobsoni in Luzon Baetidae, with 11 species from five genera; Caenidae, with along with Sroka & Soldán’s (2008) described yet unnamed four species from two genera; Heptageniidae, with 16 species Sparsorythus sp. 4 from Mt. Apo of Mindanao Island are from six genera; Prosopistomatidae, with two species from a the only two currently recognized tricorythids in the single genus; and one species each for Ephemeridae, Lepto- Philippines. phlebiidae, Teloganodidae, and Tricorythidae. Based on the newly collected materials from Layawan From a recent survey of aquatic insects conducted in River of Mt. Malindang on Mindanao Island, this study described one new mayfly species, Sparsorythus buntawensis 1 Department of Life Science, National Taiwan Normal University, No. 88, Sec. 4, Tingzhou Rd., Taipei 11677, Taiwan; sp. nov. (Tricorythidae). We associated sexes and life 2 Department of Biological Sciences, Mindanao State University – stages of conspecifics using DNA barcode (Gattolliat et al., Iligan Institute of Technology, 2015; Webb et al., 2012; Zhou et al., 2009; Ball et al., A. Bonifacio Ave., Tibanga, Iligan City 9200, Philippines; 2005). 3 Forestal Healing Homes and Therapeutic Milieu, Davao City 8000, Philippines. 4 Correspondence: [email protected]. MATERIALS AND METHODS

* Article Details Submitted : 25 April 2016 Layawan River is characterized by a rocky riverbed with

Accepted : 12 October 2016 large boulders, dense riparian vegetation upstream, and

Philippine Journal of Systematic Biology | Batucan et al.: A New Species of Mayfly (Ephemeroptera: Trichorythidae)

Figure 1: (a) The habitat of Layawan River, and (b) a modified 3D map of Mt. Malindang (ICRAF, 2013) showing the river system and sampling sites. Inset is Mindanao Island located in southern Philippines, the region in red is the location of Mt. Malindang Range. farmlands downstream (Fig. 1a). This river drains into the minute of extension at 72 ºC ( 35 cycles), and 10 minutes Oroquieta watershed and is one of the major rivers in the Mt. of final extension at 72 ºC. PCR products were visualized Malindang Range Natural Park (Hansel et al., 2006), the only on 1.5% agarose gels and purified by shrimp alkaline representative natural forest remaining in the Zamboanga phosphatase/exonuclease I, and then subjected to DNA Peninsula biogeographic zone of Mindanao Island (Arances, sequencing using the BigDye® terminator 3.1 sequencing 2006) and newly designated as an ASEAN Heritage Park. kit on an ABI 3730XL DNA Analyzer. The region where the river lies has a Type II Philippine climate characterized by no dry season with very pronounced The forward and reverse COI sequences were assembled rainy season from December to February (PAGASA, 2015). using Seqman v7.1.0 (DNASTAR, Madison, USA). All Ten sites (Fig. 1b) ranging from 73 to 1,215 m elevation along metadata and sequences >600 bps were uploaded to the the stretch of Layawan River were sampled. Two sampling Barcode of Life Database (BOLD, www.boldsystems.org) efforts were conducted, October 1–6, 2013 and April 13– 18, and submitted to GenBank (Table 1). Sequence analyses 2014. Nymphs were collected manually while adults were were carried out using MEGA 6 (Tamura et al.., 2013). collected through LED light traps. All field–collected Pairwise distances were calculated using Kimura–2– specimens were stored in 90% alcohol, then transferred to parameter model with pairwise deletion for gap handling 95% alcohol in the laboratory, and finally stored at –20 ºC and 1000 bootstrap replications to determine the genetic freezers. Nymphal specimens were carefully dissected and distance between conspecific individuals. slide–mounted using Bioquip Euparal mounting medium. Morphological observations and imagery were made using a Nikon EOS700d digital camera mounted on an Olympus TAXONOMY SZ61 stereomicroscope with 10 to 40x magnification. From the images captured, measurements of mayfly body parts Order Ephemeroptera were made using ImageJ v1.46r software (National Institutes Family Tricorythidae Lestage, 1942 of Health, Bethesda, MD, USA). Genus Sparsorythus Sroka & Soldan, 2008 Type species Sparsorythus bifurcatus Genomic DNA was extracted from the legs of the insects Sroka & Soldan, 2008 using a standard phenol–chloroform protocol (Lin & Wood, 2002). To amplify the fragments of cytochrome c oxidase subunit I (COI) gene of animal DNA barcode, the forward Sparsorythus buntawensis Batucan, Nuñeza, & Lin sp. nov. LCO1490 and reverse HCO2198 primer (Folmer et al., 1994) (Figs. 2–17). were used for the Polymerase Chain Reaction (PCR). Each PCR reaction contained 1 µL of DNA, 1 µL of Taq Nymph Holotype: body generally light brown to pale yellow polymerase, 2 µL of forward and reverse primer, 4 µL of with black dorsal and ventral markings (Fig. 2). Margins of dNTPs, 5 µL of buffer, and 35 µL of double–distilled water. head, abdomen, and legs yellow to pale yellow. Body The PCR procedure included: one minute of denaturation at length 5–6 mm. Cerci & paracercus 1.2 and 1.3 times as 94 ºC, followed by 45 seconds of annealing at 53 ºC, one long as body.

Philippine Journal of Systematic Biology | Batucan et al.: A New Species of Mayfly (Ephemeroptera: Trichorythidae)

Figure 2: Sparsorythus buntawensis sp. nov. (a) nymph, (b) female adult, and (c) male adult. Scale ½ mm.

Head: Width to length ratio 1:1.4 (Fig. 3). Male head (Fig. 3a) width is 2:1, 2.1:1, and 2.2:1 for fore, middle, and hind legs, with median emargination posteriorly, more ovate than that of respectively. Fore femora (Fig. 6a) with black pointed setae female (Fig. 3b). Eyes black, ocelli black and smooth–edged, running halfway along depression on upper surface, triangle to bean–shaped. Compound eyes of males and directed apically and interspersed with short yellow setae, females approximately of same size. Ratio of distance upper margin with setae medially subequal in length as between compound eyes and eye width 5.2:1. upper surface setae, setal length and point variable. Ventral margin of fore tibia (Fig. 6a) with fine long hairs and one Mouthparts: Labrum (Fig. 4a) twice wider than long. stout apical pointy setae, dorsal margin with shorter and Hypopharynx (Fig. 4b) 1.3 times as wide as long with a deep thinner hairs. Dorsal surface of middle and hind femora median cleavage. Right prostheca (Fig. 4c) notched, (Fig. 6c) with very small stout setae, absent in depression. triangular, with several short pointed teeth and ½ the length of Mid- and hind femora longer than tibia. Ventral margin of inner incisor. Left prostheca (Fig. 4d) rod–like, pointed and middle tibia (Fig. 6b) with small and blunt setae, apex with notched at apex, ¾ the length of outer incisor. One stout two pointed setae. Dorsal margin of middle and hind setae near base of left prostheca, as long as right prostheca. femora (Fig. 6b, 6c) with stout and long blunt setae, Maxilla (Fig. 4e) without palps. Labial plate (Fig. 4f) without irregularly alternating in length, running medial distally. invagination at mid-anterior margin. Numerous basal setae on Dorsal margin of hind tibia (Fig. 6c) have similar setae but labial plate as long as lateral setae at second segment of more pointy ends. Caudal filament segments (Fig. 7) labial palp. surrounded with short, thin setae having maximum length approximately 1/3 of segment. Thorax and Abdomen: Gills (Fig. 5) present from segments II to VI, dorsal lamellae on segment VI (Fig. 5e) with tiny, short, Male Imago Paratype: Body length 4–5 mm. General marginal bristles. Legs (Fig. 6) robust. Length ratio (femur: coloration light black to gray. Cerci twice longer than body, tibia: tarsus): foreleg (Fig. 6a) 2.7:3:1, middle leg (Fig. 6b) paracercus approximately 2.6 times longer than body 2.9:2.7:1, hind leg (Fig. 6c) 3.2:2.8:1. Ratio of femur length to length.

Philippine Journal of Systematic Biology | Batucan et al.: A New Species of Mayfly (Ephemeroptera: Trichorythidae)

Figure 3: Nymph of S. buntawensis sp. nov. dorsal view of head (scale ¼ mm), (a) male, (b) female. Figure 4: Dorsal view of mouthparts of nymphs (scale 1/8 mm), (a) labrum, (b) hypopharynx, (c) right mandible, (d) left mandible, (e) maxilla, (f) labium. Figure 5: Ventral view of gills (scale ¼ mm), (a) II, (b) III, (c) IV, (d) V, (e) VI. Figure 6: Dorsal view of legs (scale ¼ mm), (a) fore leg, (b) middle leg, (c) hind leg. Figure 7: Setation of caudal filament (scale 1/10 mm).

Head: Gray to black, width 1 mm (Fig. 8). Compound eyes Female Adult Paratype: Body length 4–5 mm. Cerci and approximately of same size as those of females. Ratio of paracercus 1.3 and 1.5 times as long as body length. distance between compound eyes and eye width, 5:1. Scape to pedicel ratio 1:2. Head: Gray to black, width 1.2 mm (Fig. 13). Ratio of distance between compound eyes and eye width, 3.4:1 Thorax and Abdomen: Prothorax gray to black with brown to (males 5:1), compound eyes slightly larger than those of white blotches. Mesothorax and metathorax brown, gray to males. Scape to pedicel ratio 1:2. black, darker coloration at junctions of segments. Femur gray to black, tibia and tarsus gray to brown. Cerci white, segment Thorax and Abdomen: Prothorax gray to black with brown junctions gray to black. Segments of paracercus twice the to white blotches. Meso- and metathorax brown to black. length of cerci segments (Fig. 9). Segment I of forceps (Fig. Femur gray to black, tibia and tarsus white to brown. Cau- 10) approximately 1/3 of segment II, penis lobe constricted dal filaments covered with short, fine setae (Fig. 14). Suba- subapically, slightly extending beyond segment I of forceps. nal plate slightly pointed (Fig. 15). Venation of forewing Forewing venation gray to black, median to posterior surface (Fig. 16) similar to that of male, wing surface translucent. transparent (Fig. 11). Leg ratio (femur: tibia: tarsus): foreleg Forewing 6.5 mm long, surface of wing dark-colored anteri- (Fig. 12a) 2.3:2.12:1, middle leg (Fig. 12b) 2.6:2.2:1, hind leg orly and lighter posteriorly, with microtrichia. Legs (Fig. 17) (Fig. 12c) 3.6:3.3:1. Claws ephemeroid. equipped with one hooked claw and one pointy claw,

Philippine Journal of Systematic Biology | Batucan et al.: A New Species of Mayfly (Ephemeroptera: Trichorythidae)

Philippine Journal of Systematic Biology | Batucan et al.: A New Species of Mayfly (Ephemeroptera: Trichorythidae) pointy claw, femora wider and longer than those of males, leg Diagnosis and Discussion: The nymph of S. buntawensis ratio (femur: tibia: tarsus): foreleg (Fig. 17a) 3.3:4:1, middle sp. nov. differ from known Oriental tricorythids mainly leg (Fig. 17b) 3.7:3.3:1, hind leg (Fig. 17c) 5.7:5.2:1. through the deeper and smoother cleft of its hypopharynx, the longer than wide superlinguae, the highest ratio of Life Stage Association: Two COI barcode sequences of 641 distance between compound eyes and eye width, and the bps for each life stage were obtained. Mean intraspecific K2P largest leg length ratio. S. buntawensis is closest to S. sequence divergence between nymphs and adults of S. grandis in terms of leg length ratio and the absence of buntawensis sp. nov. was 10.2 ± 1.0% (n=4). Within the life medial rill on the hypophrarynx. However S. grandis has a stages, sequence divergence was 3.6 ± 0.7% (n=2) for more oblongate hypopharynx, shorter and wider nymphs and 0.6 ± 0.3% (n=2) for adults. No COI barcode se- superlinguae, and narrower and more pointy cleft whereas quences of other tricorythid species are currently available in S. buntawensis is not oblongate, has deeper, wider, and Genbank and BOLD for comparison. Nevertheless, the aver- blunt cleft, and a superlinguae with pronounced concavity age intraspecific sequence divergence of COI in S. buntawen- in its inner anterior edge. The shared morphological sis sp. nov. (10.2%) is much lower than the interspecific diver- characters between S. buntawensis sp. nov. and S. gence (18%) for all mayfly species (13.7–25.8% for Tricorythi- ceylonicus are the similar length of setae of caudal dae’s sister families, Leptohyphidae and Ephemerellidae; Ball filaments, middle & hind femora, and eye to eye width ratio. et al., 2005), this suggests that these specimens likely belong Although Ulmer (1924) recorded imagoes of S. jacobsoni to the same species. Also, specimens were collected at the from the Philippines, there is no other record of S. same site and season, and no other tricorythid species were jacobsoni being found in other Philippine islands and no found in the area. type specimens were available for access by the authors. Based on Ulmer’s (1913, 1924) and Sroka & Soldan’s Etymology: The species is named after Buntawan, the locality (2008) description, S. jacobsoni is generally larger than S. where the species was first found. buntawensis sp. nov. It also has a small nick in the median anterior margin of its labial plate whereas this is absent in Type Designation and Repository: Holotype: 1 nymph (parts S. buntawensis sp. nov. In comparison with other on slides), PNM 13547, Layawan River, Upper Toliyok, Oro- Sparsorythus, S. buntawensis sp. nov. has a more quieta City, Philippines, elevation 185 m, 3 October 2013, 08° constricted penial lobe. 25'25.0788" N, 123°41'48.3612" E. Paratypes: 6 imagoes (in 95% alcohol), PNM 13548, PNM 13549, PNM 13550, PNM The adults of S. buntawensis sp. nov., exhibit a marked 13551, PNM13552), Layawan River, Buntawan, Oroquieta sexual dimorphism in head size, in which males have City, Philippines, elevation 73 m, 6 October 2013, 08° smaller heads than do females. For the female of S. 27'17.3988" N, 123°44'41.8200" E; PNM 13553, Layawan buntawensis sp. nov., its paracercus is shorter than the River, Lower Tuminawan, Oroquieta City, Philippines, eleva- cerci. After the final molt to adult, in contrast to S. tion 610 m, 17 April 2014 08°23'05.5572" N, 123°39'01.4220" celebensis, S. buntawensis sp. nov. females have E; 5 nymphs (in 95% alcohol), PNM 13554, PNM 13555, NM prominent microtrichia on the fore wings and its forelegs 13556, PNM 13557, Layawan River, Lower Toliyok, Oroquieta are equipped with two claws wherein one is hooked and City, Philippines, elevation 161 m, 08°25'58.2600" N, 123° the other one is pointy. Coupled with the absence of usual 42'29.5200" E; PNM 13558, Layawan River, Upper Toliyok, imaginal characteristics of the females and absence of Oroquieta City, Philippines, elevation 185 m, 08°25'25.0788" imaginal females despite the intensive sampling, this obser- N, 123°41'48.3612" E; deposited along with the holotype in vation leads us to conclude that the females of S. the National Museum of Natural History, Philippine National buntawensis sp. nov. have only one adult stage as com- Museum, Manila, Philippines. All types were collected and pared to two stages in males. This non-molting female subi- prepared by Leocris S. Batucan Jr.. mago is similar to the confirmed non-molting Tricorythidae such as Tricorythus varicauda, T. discolor, and T. tinctus (Kluge, 2010). Figure 8: Dorsal view of S. buntawensis sp. nov. Head of male adult (scale: 1/16 mm). Figure 9: Male caudal filaments (dorsal, scale: ¼ mm). ACKNOWLEDGEMENTS Figure 10: Penis (ventral, scale: 1/16 mm). Figure 11: Forewing of male (scale ½ mm). We thank the Advanced Science and Technology Human Figure 12: Legs of male imago (scale: ¼ mm Resource Development Program (ASTHRDP) of the Figure 13: S. buntawensis sp. nov. head of female adult Department of Science and Technology (DOST) of the (scale: 1/16 mm). Philippines for the funding, and Manila Economic and Figure 14: Female caudal filaments (dorsal, scale: ¼ mm). Cultural Office–Taiwan Economic and Cultural Office Figure 15: Subgenital plate (ventral, scale: ¼ mm). Sandwich Scholarship Program (MECO–TECO SSP) for Figure 16: Female forewing (scale ½ mm). the scholarship to LJB. We also thank the City Figure 17: Legs of adult females (scale: ¼ mm).

Philippine Journal of Systematic Biology | Batucan et al.: A New Species of Mayfly (Ephemeroptera: Trichorythidae)

Environmental and Natural Resources Office (CENRO) of Oroquieta for providing guides during sampling, and the

Department of Environment and Natural Resources’

GenBank Accession KT25014 1 KT25014 2 KT25014 3 KT25014 4

Protected Area Management Board Region X for issuing a

15 15 15 15

gratuitous permit (GP No. R102010–04). Gratitude is

- - - - extended to the members of the Systematics and Evolutionary Biology Laboratory of NTNU, and to Pavel Sroka

for giving valuable comments regarding the Tricorythidae. We

LYWN012 LYWN011 LYWN023 LYWN009 ProcessID also thank the two anonymous reviewers who made efforts in improving this manuscript and Robert Coombs of ICRAF for providing us a 3D map Mt. Malindang. This research was supported by the Ministry of Science and Technology of

Taiwan (NSC 100–2311–B–029–004–MY3 & MOST 103–2311

–B–029–001–MY3 to CPL). The authors acknowledge the

National Core Facility Program for Biotechnology, Taiwan

(MOST 103–2319–B–010–001) for DNA sequencing.

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Philippine Journal of Systematic Biology | Batucan et al.: A New Species of Mayfly (Ephemeroptera: Trichorythidae)

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